The long-term goal of the proposed research is to study the mechanisms responsible for age-related dege- nerative processes. The understanding of these mechanisms may help to find ways to slow down the corres- ponding processes thus moving the onset of deterioration outside the normal human lifespan. Accumulation of mtDNA mutations has been long hypothesized as a probable cause of the various symptoms of aging, however, proof of such involvement was lacking. There is no consensus regarding which tissues and/or cell types if any are most likely to be affected. Our recent research has demonstrated unprecedented accumulation of mtDNA deletions in the substantia nigra of the aged human brain, which results in respiratory chain defects in pigmented neurons. This demonstration is important conceptually as an example the most significant involvement of somatic mtDNA mutations in an age-related degenerative process, but it also raises questions regarding the health concequences of this process, and whether it is limited to s. nigra. The specific aims therefore are: (1) To test the hypothesis that clonal expansions of mtDNA mutations, either deletions or point mutations, cause functional defects in specific cell types of the brain, and/or cause degeneration or predispose cells to degeneration. (2) To test the hypothesis that accumulation of mtDNA deletions in pigmented neurons of substantia nigra contributes to the development of Mild Parkinsonian Signs in seniors. We will compare the prevalence of cells with mtDNA deletions in a collection of well- characterized brains with respect to the presence of parkinsonian signs. (3) To develop new methodologies for mtDNA mutation quantification in individual cells. The methods include mtDNA Fluorescent In Situ Hybridization (FISH) for quantification of mtDNA deletions, and a novel massive parallel 454 DNA sequencing approach for studying mtDNA point mutations. In short, we have discovered a novel, common, age-related degenerative process in the brain involving the DNA of mitochondria, the power plants of the cell. This process affects an area involved in Parkinson's disease. We are therefore exploring if the process is responsible for movement problems widespread among the senior population. We are also testing whether similar processes affect other parts of the aging brain.